Microorganisms with increased photosynthetic capacity

ABSTRACT

Microorganisms with increased photosynthetic capacity are described. Increased photosynthetic capacity is achieved by down-regulating activity of the RpaB pathway. The microorganisms include Cyanobacteria, including genetically-modified Cyanobacteria.

CROSS REFERENCE TO RELATED APPLICATION

This Application is a continuation of the U.S. Non-Provisional patentapplication Ser. No. 15/569,022 filed Oct. 24, 2017, now U.S. Pat. No.10,654,901, which is a U.S. National Stage Entry of PCT/US16/28785 filedApr. 22, 2016 which claims priority to U.S. Provisional PatentApplication No. 62/152,506 filed Apr. 24, 2015, the entire contents ofwhich are incorporated by reference herein.

STATEMENT REGARDING SEQUENCE LISTING

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: a computer readableformat copy of the sequence listing (filename:LUBI-003/02US_SubSeqList_ST25.txt, date recorded: Sep. 3, 2020, filesize 62.1 kilobytes).

FIELD OF THE DISCLOSURE

The disclosure provides microorganisms with increased photosyntheticcapacity. Increased photosynthetic capacity is achieved bydown-regulating activity of the RpaB pathway. The microorganisms includeCyanobacteria, including genetically-modified Cyanobacteria.

BACKGROUND OF THE DISCLOSURE

Photosynthesis is a process by which solar energy is converted intochemical bond energy. The process of photosynthesis ultimately resultsin biomass accumulation. Biomass can be used to produce energy, fuel,chemicals, and food. As examples, bioethanol can be produced throughalcohol fermentation of saccharified carbohydrate, and biodiesel oil andbiojetfuel can be produced from neutral lipids such as waxesters andtriglycerides. Further, photosynthesis processes environmental carbondioxide.

Photosynthetic crops such as soy beans, corn, and palms have been usedas raw materials to produce biofuel and other products. Use of ediblecrops for such purposes, however, can contribute to food shortages.Non-edible crops such as jatropha and camelina have also been used, butthese crops have low yields per unit area.

Photosynthetic microorganisms similarly can be cultivated to produceenergy, fuel, chemicals, and food, as well as to process environmentalcarbon dioxide. In fact, many of these photosynthetic microorganisms arecapable of producing larger amount of oils, fats and carbohydrates thanplants.

SUMMARY OF THE DISCLOSURE

The present disclosure provides modified photosynthetic microorganismswith increased photosynthetic capacity. Increased photosyntheticcapacity is achieved by down-regulating activity of the RpaB pathway.Increased photosynthetic capacity can increase total carbon fixation,production of carbon containing compounds, and growth (biomassaccumulation), among other uses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts relative mRNA expression levels of hliA in wild type(e.g., non-modified) and mutant strain MX1296 (Ptrc:N-rpaB) without andwith addition of 1 mM IPTG.

FIG. 2 depicts total photosystem II (PSII) activity of wild type andstrain MX1296 grown without or with 1 mM IPTG in medium. Activity wasmeasured by determining the rate of oxygen evolution of whole cells inthe presence of para-benzoquinone and potassium ferricyanide, whichserve to accept electrons directly from PSII, allowing for PSII oxygenevolution to run at maximal rate, independent of down-stream proteins inthe electron transport chain.

FIG. 3 depicts total electron transport chain activity of wild type andstrain MX1296 grown with 1 mM IPTG in medium. Activity was measured bydetermining the rate of oxygen uptake of whole cells in the presence ofmethyl viologen and potassium cyanide, which serve to accept electronsdirectly from photosystem I (PSI), allowing for the entire electrontransport chain to run at maximal rate, independent of down-streamproteins in, e.g., carbon fixation or nitrate reduction.

FIG. 4 depicts total photosystem II (PSII) activity of wild type, strainMX1504 (N-RpaB) with 1 mM IPTG in medium, and strain MX2335 (N-SrrA,without added IPTG). Activity was measured by determining the rate ofoxygen evolution of whole cells in the presence of para-benzoquinone andpotassium ferricyanide, as described in relation to FIG. 2. MX1504 is anN-RpaB strain built in a different WT background than MX1296. Theappropriate WT control for MX1296 is named TGA1-75 where the appropriateWT control for MX1504 is named TGA1-30. This FIG. indicates Ptrc_N-SrrAis effective without induction with IPTG, whereas Ptrc_N-RpaB is moreeffective with IPTG.

FIG. 5 depicts growth, as measured by optical density at 750 nm (OD750)of wild type, MX2335, and MX1504 with and without 1 mM IPTG afterrepeated dilutions with fresh media. Arrows indicate time points atwhich sample aliquots were removed for dry weight determination, whichis given in FIG. 6.

FIG. 6 depicts growth yields of WT, MX1504 without and with 1 mM IPTG,and MX2335 as determined 96 and 144 hours into the experiment. Thisgrowth experiment is the same as that for which data in FIG. 5 ispresented. FIGS. 5 and 6 show that photoautotrophic growth ofphotosynthetic microorganism with down-regulated RpaB pathway activityis increased by repeatedly replacing a volume fraction of the liquidculture with an equivalent volume of new growth media.

FIG. 7 provides a scheme depicting multiple routes through which RpaBmay affect expression of light responsive genes. Overexpression offull-length RpaB may increase the amount of full length, phosphorylatedRpaB, which acts as an activator for some genes and a repressor forothers. Dephosphorylated RpaB may as act as an activator or repressorfor other sets of genes.

FIG. 8A-J provides exemplary sequences referenced throughout thedisclosure.

DETAILED DESCRIPTION

Photosynthesis is a process by which solar energy is converted intochemical bond energy. The overall reaction of photosynthesis is thelight-driven conversion of carbon dioxide and water to glucose andoxygen:6CO₂+6H₂O→C₆H₁₂O₆+6O₂Photosynthesis is observed in plants as well as in bacteria, andblue-green algae.

The process of photosynthesis ultimately results in biomassaccumulation. Biomass can be used to produce energy, fuel, chemicals,and food. As examples, bioethanol can be produced through alcoholfermentation of saccharified carbohydrate, and biodiesel oil andbiojetfuel can be produced from neutral lipids such as waxesters andtriglycerides. Further, photosynthesis processes environmental carbondioxide.

Photosynthesis includes two stages called the light reactions and thedark reactions. The light reactions require the presence of light, whilethe dark reactions do not depend on direct light exposure. In the lightreactions, sunlight is absorbed and drives an electron transport chainthat results in the formation of the energy carriers NADPH and ATP,forming O₂ as a by-product. In the dark reactions, a reaction driven byNADPH and ATP reduces CO₂ to glucose.

Photosystems are large multiprotein complexes that allow, incollaboration with other components, the conversion of captured solarenergy into chemical bond energy via the electron transport chain. Ingeneral, photosystems are made up of two components: (1) a photochemicalreaction center that allows solar energy to be converted into chemicalenergy, and (2) an antenna complex which captures light energy andtransfers it to the photochemical reaction center, resulting inexcitation of the photosystem.

The source of electron replenishment in a photosynthesis system differsaccording to the reaction center type. In purple non-sulfur bacteria,for example, electrons are cycled back to the reaction center bywater-soluble electron carriers, for example, a cytochrome c typeprotein. In oxygenic photosynthetic organisms, including Cyanobacteria,red and green algae and plants, electron flow is non-cyclic, and occursin two steps that involve two photosystems: Photosystem I (PSI) andPhotosystem II (PSII). In these types of reactions, the deficit ofelectrons can be replenished by electrons taken from water molecules.

PSII is a complex composed of proteins, pigments and cofactors, locatedwithin thylakoid membranes. PSII splits water into oxygen, protons andelectrons. Oxygen is released into the atmosphere and is responsible formaintaining aerobic life on Earth. The electrons are immediatelyenergized by a photon (λ=680 nm) in PSII and passed from one compound toanother, all of which compose the electron transport chain. Most of theelectron carriers are quinones (Q), plastiquinones (PQ), or cytochromes(Cyt).

More particularly, the process of electron transfer in PSII includes thefollowing steps: upon illumination, a P₆₈₀ chlorophyll is photoexcited.The photoexcited P₆₈₀ transfers electrons via intermediate cofactorscalled pheophytin a and plastoquinone A (PQ, Q_(A)) in order to finallydoubly reduce a transiently bound PQ molecule (Q_(B)). Q_(B) ²⁻ isprotonated and released from the reaction center into the thylakoidmembrane. The redox active cofactors that enable electron transfer fromwater to the secondary quinone acceptor Q_(B) are mainly embedded withintwo proteins called D1 and D2. Under normal conditions of illumination,the D1 protein of the reaction center core is irreversibly damaged overtime and is replaced in a fashion that preserves the integrity of thePSII complex.

A second input of light energy (λ=700 nm) occurs during PSI and theenergized electrons are passed to the terminal electron carrier,ferredoxin (Fd). Reduced Fd can serve as an electron donor to theferredoxin-NADP*-reductase (FNR) enzyme. In a parallel process(photophosphorylation), H⁺ are released where they generate a H⁺gradient that is used to drive ATP production via ATP synthase. NADPHand ATP are subsequently used to produce starch and other forms ofenergy storage biomass.

Cyanobacteria are the only group of organisms that are able to reducenitrogen and carbon in aerobic conditions. The water-oxidizingphotosynthesis is accomplished by coupling the activity of PSII and PSI(the Z-scheme). In anaerobic conditions, Cyanobacteria are also able touse only PSI (i.e., cyclic photophosphorylation) with electron donorsother than water (e.g., hydrogen sulfide, thiosulphate, or molecularhydrogen), similar to purple photosynthetic bacteria. Furthermore,Cyanobacteria share an archaeal property—the ability to reduce elementalsulfur by anaerobic respiration in the dark. The Cyanobacterialphotosynthetic electron transport system shares the same compartment asthe components of respiratory electron transport. Typically, the plasmamembrane contains only components of the respiratory chain, while thethylakoid membrane hosts both respiratory and photosynthetic electrontransport.

Phycobilisomes are complexes of phycobiliproteins and colorlesspolypeptides which function as the major light harvesting antennae inblue-green and red algae. The phycobilisome components(phycobiliproteins) are responsible for the blue-green pigmentation ofmost Cyanobacteria. Color variations are mainly due to carotenoids andphycoerythrins, which may provide the cells with a red-brownishcoloration. In some Cyanobacteria, the color of light influences thecomposition of phycobilisomes. In green light, the cells accumulate morephycoerythrin, whereas in red light they produce more phycocyanin. Thus,the bacteria appear green in red light and red in green light. Thisprocess is known as complementary chromatic adaptation and represents away for the cells to maximize the use of available light forphotosynthesis.

As suggested, photosynthetic organisms must cope with environmentalchanges in their habitats, such as fluctuations in incident light.Changes in light quantity or quality (i.e., spectral composition) canresult in imbalanced excitation of PSII and PSI and decrease theefficiency of photosynthetic light reactions. Photosynthetic organismscan counteract such excitation imbalances with changes in geneexpression.

OmpR response regulators are response regulators wherein theirphosphorylation promotes specific DNA binding by enhancing dimer oroligomer formation. In some cases, dephosphorylated OmpR responseregulators have >10-fold lower affinity to their binding sites thanphosphorylated forms. RpaA and RpaB are two types of OmpR responseregulators.

The NblS kinase (NblS)-RpaB signaling pathway is the most conservedtwo-component system in Cyanobacteria. This pathway is involved inregulation of circadian-based changes in gene expression, regulation ofphotosynthesis, and acclimatization to a variety of environmentalconditions.

The full length protein RpaB has an N-terminal phospho-receiver domainand a C-terminal DNA binding domain. The C-terminal domain isresponsible for binding promoter regions such as HLR1 (highlight-responsive element 1) and repressing transcription of downstreamgenes when RpaB is phosphorylated (at the N-terminal side of theprotein). N-terminal fragments of the RpaB protein can have aphospho-receiver domain but no known DNA binding domain. Thephosphorylatable residue of RpaB is thought to be Asp56.

With regard to regulation of circadian-based changes in gene expression,RpaB binds the KaiBC promoter and represses transcription of kaiBC andother target genes during subjective night (e.g., ˜LL0). Duringsubjective day (e.g., ˜LL4-8), RpaB is released from these promoters,likely through the effects of RpaA, to allow transcription of therepressed genes.

As stated, with regard to regulation of photosynthesis, RpaB binds tothe promoter HLR1. The HLR1 motif includes two direct repeats of SEQ IDNO: 1 separated by two nucleotides. When bound to HLR1, RpaB repressestranscription of genes, such as rpoD3 and hliA. Under high light stress,RpaB is dephosphorylated in a process mediated by NblS to allowtranslation of these genes.

Decreasing the copy number of RpaB genes in Cyanobacteria decreasesenergy transfer from phycobilisomes to PSII and increases energytransfer from phycobilisomes to PSI. Thus, it is been suggested thatRpaA and RpaB regulate expression of proteins involved in the couplingof phycobilisomes to PSI or PSII. With regard to acclimation to otherenvironmental conditions, RpaB has been shown to modulate transcriptionof genes in response to cold shock as well as osmotic, salt andoxidative stresses. In spite of the importance of the NblS-RpaBsignaling pathway, actual input signals and output responses remainlargely unknown.

SrrA is homologous to RpaB, but has distinctly different regulatoryroles in Synechococcus elongatus PCC 7942 and is coded by anon-essential gene. SrrA is also known as Crr71. RpaB and SrrA are theonly known substrates of the NblS kinase.

The current disclosure provides microorganisms with increasedphotosynthetic capacity. Increased photosynthetic capacity can beachieved by down-regulating activity of the RpaB pathway. Numerousmechanisms to down-regulate activity of the RpaB pathway are describedherein. Particular examples include expression of RpaB decoys,expression of SrrA decoys and/or direct down-regulation of NblS,through, for example, CRISPRi.

Aspects of the current disclosure are now described in more detail.

Photosynthetic Microorganisms. Photosynthetic microorganisms of thedisclosure may be any type of organism capable of performingphotosynthesis wherein the microorganism has been modified to havedown-regulated RpaB pathway activity.

Exemplary photosynthetic microorganisms that are either naturallyphotosynthetic or can be engineered to be photosynthetic includebacteria (e.g., Cyanobacteria); fungi; archaea; protists; eukaryotes,such as a green algae; and animals such as plankton, planarian, andamoeba. Examples of naturally occurring photosynthetic microorganismsinclude Arthrospira (Spirulina) maxima, Arthrospira (Spirulina)platensis, Dunaliella salina, Botycoccus braunii, Chloella vulgaris,Chlorella pyrenoidosa, Serenastrum capricomutum, Scenedesmusauadricauda, Porphyridium cruentum, Scenedesmus acutus, Dunaliella sp.,Scenedesmus obliquus, Anabaenopsis, Aulosira, Cylindrospermum,Synechoccus sp., Synechocystis sp., Cyanobacterium aponinum, andTolypothrix sp.

Cyanobacteria, also known as blue-green algae, blue-green bacteria, orCyanophyta, is a phylum of bacteria that obtain their energy throughphotosynthesis. As stated, Cyanobacteria can produce metabolites, suchas carbohydrates, proteins, lipids and nucleic acids, from CO₂, water,inorganic salts and light. Any Cyanobacteria may be used according tothe disclosure. In particular embodiments the Cyanobacteria must begenetically manipulatable, e.g., permissible to the introduction andexpression of exogenous genetic material (e.g., exogenous nucleotidesequences).

Cyanobacteria include both unicellular and colonial species. Coloniesmay form filaments, sheets or even hollow balls. Some filamentouscolonies show the ability to differentiate into several different celltypes, such as vegetative cells, the normal, photosynthetic cells thatare formed under favorable growing conditions; akinetes, theclimate-resistant spores that may form when environmental conditionsbecome harsh; and thick-walled heterocysts, which contain the enzymenitrogenase, vital for nitrogen fixation.

Examples of Cyanobacteria that may be utilized and/or geneticallymodified according to the methods described herein include ChroococcalesCyanobacteria from the genera Arthrospira, Aphanocapsa, Aphanothece,Chamaesiphon, Chroococcus, Chroogloeocystis, Coelosphaenum,Crocosphaera, Cyanobacterum, Cyanobium, Cyanodictyon, Cyanosarcina,Cyanothece, Dactylococcopsis, Gloecapsa, Gloeothece, Merismopedia,Microcystis, Radiocystis, Rhabdoderma, Snowella, Synychococcus,Synechocystis, Thermosenechococcus, and Woronichinia; NostacalesCyanobacteria from the genera Anabaena, Anabaenopsis, Aphanizomenon,Aulosira, Calothrix, Coleodesmium, Cyanospira, Cylindrospermosis,Cylindrospermum, Fremyella, Gleotrichia, Microchaete, Nodularia, Nostoc,Rexia, Richelia, Scytonema, Sprirestis, and Toypothrix; OscillatorialesCyanobacteria from the genera Arthrospira, Geitlerinema, Halomicronema,Halospirulina, Katagnymene, Leptolyngbya, Limnothrix, Lyngbya,Microcoleus, Oscillatoria, Phormidium, Planktothricoides, Planktothrix,Plectonema, Pseudoanabaena/Limnothrix, Schizothix, Symploca,Trichodesmium, and Tychonema; Pleurocapsales Cyanobacteria from thegenera Chroococcidiopsis, Dermocarpa, Dermocarpella, Myxosarcina,Pleurocapsa, Stanieria, and Xenococcus; Prochlorophytes Cyanobacteriafrom the genera Prochloron, Prochlorococcus, and Prochlorothrix; andStigonematales Cyanobacteria from the genera Capsosira, Chlorogeoepsis,Fischerella, Hapalosiphon, Mastigocladopsis, Nostochopsis, Stigonema,Symphyonema, Symphonemopsis, Umezakia, and Westiellopsis. In particularembodiments, the Cyanobacteria is from the genus Synechococcus,including Synechococcus bigranulatus, Synechococcus elongatus,Synechococcus leopoliensis, Synechococcus lividus, Synechococcusnidulans, and Synechococcus rubescens. CyanobacteriaThermosynechococcus, and Gloeobacter can also be used.

More particular embodiments include or utilize Anabaena sp. strain PCC7120, Synechocystis sp. strain PCC 6803, Nostoc muscorum, Nostocellipsosporum, or Nostoc sp. strain PCC 7120. In particular embodiments,the Cyanobacteria is Synechococcus elongatus sp. strain PCC 7942.Additional examples of Cyanobacteria that may utilized includeSynechococcus sp. strains WH7803, WH8102, WH8103 (typically geneticallymodified by conjugation), Baeocyte-forming Chroococcidiopsis spp.(typically modified by conjugation/electroporation),non-heterocyst-forming filamentous strains Planktothrix sp., Plectonemaboryanum M101 (typically modified by electroporation),Heterocyst-forming Anabaena sp. ATCC 29413 (typically modified byconjugation), Tolypothrix sp. strain PCC 7601 (typically modified byconjugation/electroporation) and Nostoc punctiforme strain ATCC 29133(typically modified by conjugation/electroporation).

In particular embodiments, the Cyanobacteria may be, e.g., a marine formof Cyanobacteria or a fresh water form of Cyanobacteria. Examples ofmarine forms of Cyanobacteria include Synechococcus WH8102,Synechococcus RCC307, Synechococcus NKBG 15041c, and Trichodesmium.Examples of fresh water forms of Cyanobacteria include S. elongatus PCC7942, Synechocystis PCC6803, Plectonema boryanum, Cyanobacterumaponinum, and Anabaena sp.

In other embodiments, a genetically modified Cyanobacteria may becapable of growing in brackish or salt water. When using a fresh waterform of Cyanobacteria, the overall net cost of their use will depend onboth the nutrients required to grow the culture and the price forfreshwater. One can foresee freshwater being a limited resource in thefuture, and in that case it would be more cost effective to find analternative to freshwater. Two such alternatives include: (1) the use ofwaste water from treatment plants; and (2) the use of salt or brackishwater.

Salt water in the oceans can range in salinity between 3.1% and 3.8%,the average being 3.5%, and this is mostly, but not entirely, made up ofsodium chloride (NaCl) ions. Brackish water, on the other hand, has moresalinity than freshwater, but not as much as seawater. Brackish watercontains between 0.5% and 3% salinity, and thus includes a large rangeof salinity regimes and is therefore not precisely defined. Waste wateris any water that has undergone human influence. It includes liquidwaste released from domestic and commercial properties, industry, and/oragriculture and can encompass a wide range of possible contaminants atvarying concentrations.

There is a broad distribution of Cyanobacteria in the oceans, withSynechococcus filling just one niche. Specifically, Synechococcus sp.PCC 7002 (formerly known as Agmenellum quadruplicatum strain PR-6) growsin brackish water, is unicellular and has an optimal growing temperatureof 38° C. While this strain is well suited to grow in conditions of highsalt, it will grow slowly in freshwater. In particular embodiments, thedisclosure includes the use of a Cyanobacteria PCC 7942, altered in away that allows for growth in either waste water or salt/brackish water.A Synechococcus elongatus PCC 7942 mutant resistant to sodium chloridestress has been described (Bagchi et al., Photosynth Res., 2007;92:87-101), and a genetically modified S. elongatus PCC 7942 tolerant ofgrowth in salt water has been described (Waditee et al., PNAS, 2002;99:4109-4114). Salt water tolerant Cyanobacteria may also be prepared asdescribed in the Examples of U.S. Pat. No. 8,394,614. According to thedisclosure a salt water tolerant strain is capable of growing in wateror media having a salinity in the range of 0.5% to 4.0% salinity,although it is not necessarily capable of growing in all salinitiesencompassed by this range. In particular embodiments, a salt tolerantstrain is capable of growth in water or media having a salinity in therange of 1.0% to 2.0% salinity. In particular embodiments, a salt watertolerant strain is capable of growth in water or media having a salinityin the range of 2.0% to 3.0% salinity.

Down-regulating activity of the RpaB pathway can be achieved throughvarious mechanisms. Down-regulation of the RpaB pathway can be achievedby, for example, decreasing the presence or activity of a protein orgene in the pathway that promotes pathway activation (e.g., full lengthRpaB, SrrA or NblS). Down-regulation of the RpaB pathway can also beachieved by, for example, increasing the presence or activity of aprotein or gene in the pathway that inhibits pathway activation (e.g., apathway phosphatase or a pathway decoy that dampens effective activityof other active pathway members (e.g., a wild-type RpaB or SrrA sequencewith the phospho-receiver domain substituted with amino acids that arenot phosphorylatable (a non-conservative substitution) or inhibitoryCRISPRi expression products).

A decrease in presence or activity of a protein or gene in a pathway canbe caused by, for example, reduction of a gene's copy number, insertionof a foreign set of base pairs into a gene (e.g., into a coding region),deletion of any portion of the gene (e.g., of all or part of a codingregion), substitution of base pairs within the gene (e.g., into a codingregion), interference with an encoded RNA transcript, the presence ofantisense sequences that interfere with transcription or translation ofthe gene; translation of an incomplete protein; incorrect folding of aprotein; expression of an unstable protein; reduced transcription of agene; incomplete transcription of a gene, or by any other activityresulting in reduced presence, expression or activity of a protein inthe pathway that promotes pathway activation.

An increase in presence or activity of a protein or gene in a pathwaycan be caused by, for example, an increase in a gene's copy number,introduction of a strong and/or inducible promoter, mechanisms toprevent degradation of encoding nucleotides or expressed proteins, orother mechanisms.

In particular embodiments, the RpaB pathway is down-regulated byexpressing an RpaB decoy and/or an SrrA decoy. Without being bound bytheory, expressed decoys will compete with full length wild type RpaBand/or Srra for phosphorylation (e.g., on Asp56 or Asp64, respectively).This competition will result in a net decrease in the phosphorylation ofwild type RpaB proteins, and thus up-regulation of RpaB-regulated geneexpression. This approach can be referred to as a “dominant interfering”phenotype as the modified photosynthetic microorganism is expected tohave a lower degree of transcriptional repression at HLR1. In otherwords, a constitutive “high light” (or deprivation of certain nutrient)phenotype can be created. Thus, in particular embodiments,down-regulation of the RpaB pathway can be evidenced by theup-regulation of an RpaB-regulated gene, such as hliA (see FIG. 1) andRpoD3.

In particular embodiments, the RpaB and/or SrrA decoy is a protein thatwill compete with wild-type RpaB for phosphorylation. The term“wild-type” can be used interchangeably with “naturally occurring” andrefers to a gene or gene product (e.g., transcript or protein) that hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild type gene or gene product is thatwhich is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the gene orgene product.

In particular embodiments, the RpaB decoy is a protein that can bephosphorylated by one or more kinases capable of phosphorylatingwild-type RpaB. In particular embodiments, the RpaB decoy is a proteinthat can be phosphorylated by NblS. In particular embodiments, the RpaBdecoy includes a wild-type RpaB phospho-receiver domain. In particularembodiments, the RpaB decoy includes a wild-type RpaB phospho-receiverdomain and 1, 2, 3, 4, or 5 wild-type amino acid residues flanking thisposition. In these embodiments, the phospho-receiver domain includesAsp56. In particular embodiments, the RpaB decoy includes a wild-typeRpaB phospho-receiver domain and does not include a wild-type DNAbinding domain or includes a non-functional DNA binding domain. Inparticular embodiments, the RpaB decoy includes an N-terminal fragmentof the wild-type RpaB, including the wild-type phospho-receiver domainbut does not include a DNA binding domain or includes a non-functionalDNA binding domain. In particular embodiments, the wild-typephospho-receiver domain includes Asp56. As indicated below, this Asp canbe replaced with phospho-receiver domain conservative substitutions suchas Glu, Ser and Thr. In particular embodiments, the RpaB decoy can beN-RpaB (SEQ ID NO: 2).

In particular embodiments, a gene from Synechococcus elongatus PCC 7942that encodes a RpaB decoy can be placed behind an inducible promoter ina neutral site (e.g., NS1) to drive expression of the RpaB decoy. Inparticular embodiments, the gene can contain the first 378 base pairs ofthe gene Synpcc7942_1453 (full length gene is 735 base pairs), followedby a stop codon, and can be placed behind an IPTG inducible promoter ina neutral site (e.g., NS1 or NS2) to drive expression of N-RpaB (SEQ IDNO: 2). This gene and associated nucleotide sequence is represented bySEQ ID NO: 23. In this sequence, the wild type start codon has beenmodified from TTG to ATG.

Overexpression of the N-RpaB protein fragment conferred atranscriptional response similar to one observed when cells are stressedwith, e.g. high light or deprivation of certain nutrients. For example,the mRNA expression level of the gene hliA, which is known to increaseunder high light, increases 5-fold relative to wild type or uninducedmutant levels, as shown in FIG. 1.

When the strain with this mutation is grown in the presence of 1 mMIPTG, it has a higher photosystem II activity and higher photosyntheticelectron transport capacity than wild type. Photosystem II activity ismeasured by determining the rate of oxygen evolution of whole cells inthe presence of para-benzoquinone and potassium ferricyanide, whichserve to accept electrons directly from PSII, allowing for PSII oxygenevolution to run at its maximal rate, independent of down-streamproteins in the electron transport chain. FIG. 2 shows the maximaloxygen evolution capacity of PSII in cells with induction of the RpaBprotein fragment (denoted N-RpaB) with 1 mM IPTG.

Electron transport activity is measured by determining the rate ofoxygen consumption of whole cells under bright light illumination in thepresence of methyl viologen and potassium cyanide. The magnitude of themeasured rate of oxygen uptake is equal to the total capacity of thecell's oxygen evolution rate when electrons are passed through itsentire electron transport chain via photosynthesis. This increasedphotosynthetic capacity can increase total carbon fixation, productionof carbon containing compounds, and growth (biomass accumulation).Overexpression of the same 378 base pair gene fragment under otherstrong promoters confers such an increase in electron transportactivity. More particularly, FIG. 3 shows the maximal electron transportchain activity of cells with and without induced production of N-RpaB.Without being bound by theory, the increase in PSII capacity allows forincreased total electron transport chain activity in Synechococcus.

SEQ ID NO: 24 provides a reference wild-type RpaB protein sequence andSEQ ID NO: 25 provides a reference wild-type RpaB gene sequence. Thesereference sequences are derived from Synpcc7942_1453. SEQ ID NO: 29provides a reference wild-type RpaB protein sequence and SEQ ID NO: 30provides a reference wild-type RpaB gene sequence derived fromArthrospira platensis (NIES39_K03840). SEQ ID NO: 31 provides areference wild-type RpaB protein sequence and SEQ ID NO: 32 provides areference wild-type RpaB gene sequence derived from Cyanobacteriumaponinum (WP_015219361.1). Additional homologous protein and genesequences can also serve as reference wild-type RpaB protein sequencesfor the purposes of this disclosure. Exemplary homologous reference RpaBprotein sequences include SEQ ID NOs: 26, 27 and 28 derived fromSynechococcus sp. (WH 8102), Tolypothrix sp. (PCC 7601), andThermosynechococcus sp. (NK55a) respectively.

In particular embodiments, the SrrA decoy is a protein that can bephosphorylated by one or more kinases capable of phosphorylatingwild-type SrrA. In particular embodiments, the SrrA decoy is a proteinthat can be phosphorylated by NblS. In particular embodiments, the SrrAdecoy includes a wild-type SrrA phospho-receiver domain. In particularembodiments, the SrrA decoy includes a wild-type SrrA phospho-receiverdomain and 1, 2, 3, 4, or 5 wild-type amino acid residues flanking thisposition. In these embodiments, the phospho-receiver domain includesAsp64. In particular embodiments, the SrrA decoy includes a wild-typeSrrA phospho-receiver domain and does not include a wild-type DNAbinding domain or includes a non-functional DNA binding domain. Inparticular embodiments, the SrrA decoy includes an N-terminal fragmentof the wild-type RpaB, including the wild-type phospho-receiver domainbut does not include a DNA binding domain or includes a non-functionalDNA binding domain. In particular embodiments, the wild-typephospho-receiver domain includes Asp64. As indicated below, this Asp canbe replaced with phospho-receiver domain conservative substitutions suchas Glu, Ser and Thr. In particular embodiments, the SrrA decoy caninclude SEQ ID NO: 34. Non-functional DNA binding domains fail to bindDNA or bind DNA, but result in a significantly reduced amount ofresulting gene expression as compared to a relevant control.

In particular embodiments, a gene from Synechococcus elongatus PCC 7942that encodes a SrrA decoy can be placed behind a promoter in a neutralsite (e.g., NS1 or NS2) to drive expression of the SrrA decoy. MX2335,Ptrc_N-SrrA, is a strain expressing the first 404 base pairs of geneSynpcc7942_2416 (SEQ ID NO; 33; full length gene is 768 base pairs (SEQID NO: 35)), followed by a stop codon, placed behind a constitutivepromoter in neutral site 2 (NS2) to drive expression of N-SrrA (SEQ IDNO: 34). In these embodiments, Ptrc is referred to as a constitutivepromoter because no addition of IPTG is necessary for low-levelinduction of expression, and low-level expression of N-SrrA issufficient to produce the decoy phenotype with increased photosyntheticcapacity (FIG. 4). The full length Synechococcus elongatus PCC 7942 SrrAis provided as SEQ ID NO: 36.

SEQ ID NO: 38 provides a reference wild-type SrrA protein sequence andSEQ ID NO: 37 provides a reference wild-type SrrA gene sequence derivedfrom Arthrospira platensis NIES-39. Additional homologous protein andgene sequences can also serve as reference wild-type SrrA sequences forthe purposes of this disclosure. An additional exemplary homologousreference SrrA protein sequences includes SEQ ID NO: 40 and anadditional exemplary homologous reference SrrA gene sequence includesSEQ ID NO: 39 derived from Procholorcoccus marinus SS120.

Based on the teachings of this disclosure, one of ordinary skill in theart can determine additional homologous sequences, relevantphospho-receiver domains, DNA binding domains, and encoding nucleotidesequences to generate functioning decoys to down-regulate RpaB pathwayactivity in photosynthetic microorganisms.

The RpaB pathway can also be down-regulated by directly down-regulatingNblS. As indicated previously, there are numerous ways to achievedown-regulation of a protein. In particular embodiments, NblS can bedown-regulated utilizing CRISPRi technology. For example, the minimalCRISPR system from Streptococcus pyogenes needed for building an NblSCRISPRi strain requires only a single gene encoding a Cas9 protein andtwo RNAs, a mature CRISPR RNA (crRNA) and a partially complementarytrans-acting RNA (tracrRNA), to target and cleave foreign DNA elementsin a sequence-specific manner. In particular embodiments, the Cas9protein can be modified by two point mutations (D10A and H841A) at theactive sites to obtain the dCas9 protein that can bind to DNA (guided bya complementary RNA sequence) without cleaving it, potentiallyinterfering with transcriptional initiation or elongation (Jinek et al.,2012, A programmable dual-RNA-guided DNA endonuclease in adaptivebacterial immunity. Science (New York, N.Y.), 337(6096), 816-21.doi:10.1126/science.1225829). Another modification can includereplacement of the two original RNAs in the native system (crRNA andtracrRNA) with an engineered small guide RNA (sgRNA) (Jinek et al.,2012; Mali et al., 2013, RNA-guided human genome engineering via Cas9.Science, 823. doi:10.1126/science.1232033).

In particular embodiments, guide RNA designed and used fortranscriptional interference of the NblS gene in Synechococcus elongatusPCC7942 can be encoded by the sequence: ttggcaacaactgcgcgata (SEQ ID NO:41) resulting in the sequence uuggcaacaacugcgcgaua (SEQ ID NO: 42). Thissequence can be expressed/placed behind a promoter such as a pTacpromoter, which is inducible by addition of 1 mM IPTG to the growthmedium. An exemplary DNA sequence to drive expression of the Cas9protein is provided as SEQ ID NO: 43 which encodes SEQ ID NO: 44. Inparticular embodiments, its expression can be driven by a promoter suchas the pBAD promoter, which is inducible by addition of 0.02% arabinoseto the growth medium.

Embodiments disclosed herein do not utilize RpaB knockouts, as completeRpaB knockout is lethal. Accordingly, embodiments disclosed hereinutilize down-regulation, rather than elimination, of RpaB pathwayactivity.

As is understood by one of ordinary skill in the art, “up-regulation”and “down-regulation” of gene and protein expression as well as RpaBpathway activity can be measured against a relevant control conditionincluding relative to the expression or activity of an unmodifiedphotosynthetic microorganism or a photosynthetic microorganism having adifferent modification (such as a modification un-related to decreasingactivity of the RpaB pathway).

In particular embodiments, conclusions are drawn based on whether ameasure is statistically significantly different or not statisticallysignificantly different from a reference level of a relevant control. Ameasure is not statistically significantly different if the differenceis within a level that would be expected to occur based on chance alone.In contrast, a statistically significant difference or increase is onethat is greater than what would be expected to occur by chance alone.Statistical significance or lack thereof can be determined by any ofvarious systems and methods used in the art. An example of a commonlyused measure of statistical significance is the p-value. The p-valuerepresents the probability of obtaining a given result equivalent to aparticular datapoint, where the datapoint is the result of random chancealone. A result is often considered significant (not random chance) at ap-value less than or equal to 0.05.

As indicated, various mechanisms to down-regulate the RpaB pathway relyon inserting exogenous nucleotide sequences into the genome of theselected photosynthetic microorganism. “Exogenous” refers to anucleotide sequence that does not naturally occur in the particularposition of the genome of the wild type photosynthetic microorganismwhere it is inserted, but is inserted at the particular position bymolecular biological techniques. Examples of exogenous nucleotidesequences include vectors, plasmids, and/or man-made nucleic acidconstructs.

As used herein, nucleotide sequences can include foreign sets of basepairs and genes encoding proteins or RNA (e.g., RpaB decoys, N-RpaB,SrrA decoys, N-SrrA, guide RNA, Cas9 or variants thereof, etc.). Inrelation to genes, this term includes various sequence polymorphisms,mutations, and/or sequence variants. In particular embodiments, thesequence polymorphisms, mutations, and/or sequence variants do notaffect the function of the encoded protein or RNA. Genes may include notonly coding sequences but also non-coding regulatory regions such aspromoters, enhancers, and termination regions. The term further caninclude all introns and other DNA sequences spliced from the mRNAtranscript, along with variants resulting from alternative splice sites.Nucleic acid sequences encoding proteins can be DNA or RNA that directsthe expression of protein or RNA. These nucleic acid sequences may be aDNA strand sequence that is transcribed into RNA or an RNA sequence thatis translated into protein. The nucleic acid sequences include both thefull-length nucleic acid sequences as well as non-full-length sequencesderived from the full-length protein or RNA. The sequences can alsoinclude degenerate codons of the native sequence or sequences that maybe introduced to provide codon preference. Thus, a gene refers to a unitof inheritance that occupies a specific locus on a chromosome andconsists of transcriptional and/or translational regulatory sequencesand/or a coding region and/or non-translated sequences (i.e., introns,5′ and 3′ untranslated sequences).

A coding sequence is any nucleotide sequence that contributes to thecode for the protein or RNA product of a gene. A non-coding sequencethus refers to any nucleic acid sequence that does not contribute to thecode for the protein or RNA product of a gene.

In addition to particular sequences provided, gene sequences to encodefor and/or interfere with proteins described herein, as well asassociated RNA are available in publicly available databases andpublications.

A “vector” is a nucleotide molecule, (e.g., a DNA molecule) derived, forexample, from a plasmid, bacteriophage, yeast or virus, into which anucleotide sequence (e.g., a gene) can be inserted or cloned. A vectorpreferably contains one or more unique restriction sites and can becapable of autonomous replication in a photosynthetic microorganism.Autonomously replicating vectors include vectors that exist asextra-chromosomal entities, the replication of which is independent ofchromosomal replication, e.g., a linear or closed circular plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. Vectors can also be integrable with the genome of thephotosynthetic microorganism. This type of vector is replicated togetherwith the chromosome(s) into which it has been integrated. Such a vectormay include specific sequences that allow recombination into aparticular, desired site of the host chromosome. Vectors used within thecurrent disclosure can include any mechanism for assuringself-replication. A vector can include a single vector (or plasmid), twoor more vectors, three or more vectors, etc. which together contain thetotal DNA required for expression of a nucleotide sequence of interestto be expressed in the photosynthetic microorganism.

As indicated, coding sequences to be expressed are operably linked to apromoter, that is, they are placed under the regulatory control of apromoter, which then controls the transcription and optionally thetranslation of the coding sequence. In the construction of heterologouspromoter/structural coding sequence combinations, it is generallypreferred to position the promoter at a distance from the codingsequence transcription start site that is approximately the same as thedistance between that a promoter and the coding sequence it controls inits natural setting. As is known in the art, some variation in thisdistance can be accommodated without loss of function. Similarly, thepreferred positioning of a regulatory sequence element with respect to acoding sequence to be placed under its control is defined by thepositioning of the element in its natural setting; i.e., the genes fromwhich it is derived.

“Constitutive promoters” are typically active, i.e., promotetranscription, under most conditions. “Inducible promoters” aretypically active only under certain conditions, such as in the presenceof a given molecule factor (e.g., IPTG) or a given environmentalcondition. In the absence of that condition, inducible promoterstypically do not allow significant or measurable levels oftranscriptional activity. For example, inducible promoters may beinduced according to temperature, pH, a hormone, a metabolite (e.g.,lactose, mannitol, an amino acid), light (e.g., wavelength specific),osmotic potential (e.g., salt induced), a heavy metal, or an antibiotic.

In particular embodiments, the promoter controlling the transcription ofthe coding sequence of interest can be a Cyanobacterial promoter. Thepromoter can be endogenous to the modified photosynthetic microorganismor can be a promoter, which was modified in order to increase itsefficiency. The promoter can also be a heterologous promoter from adifferent photosynthetic microorganism species, such as a differentCyanobacterial or bacterial species.

In particular embodiments, the coding sequence of interest is placedunder the transcriptional control of promoters (P) selected from: PaztA(e.g., from Anabaena (Nostoc) sp. strain PCC 7120); PBad; Pc1pB1; PcorT(e.g., from Synechocystis sp. PCC6803); PcrhC; PcpcB, (e.g., fromCyanobacteria ABICyano1 (SEQ ID NO: 3)); PcpcBA (e.g., from SynechocystsPCC6803); PggpS (e.g., from Cyanobacteria ABICyano1: (SEQ ID NO: 4));PhliB; PhspA; PhtpG; PisiA; PisiB; PlrtA (e.g., from CyanobacteriaABICyano1; SEQ ID NO: 5)); PnarB; PnbA (e.g., from CyanobacteriaABICyano1; (SEQ ID NO: 6)); PnirA; PntcA; PpetE; PpetJ (e.g., fromCyanobacteria ABICyano1; (SEQ ID NO: 7)); PpsbA2; PpsbD; PmrgA (e.g.,from Cyanobacteria ABICyano1; (SEQ ID NO: 8)); PnblA (e.g., from Nostocsp. PCC7120); PnirA (e.g., from Cyanobacteria ABICyano1); PnrsB (e.g.,from Synechocystis sp. PCC6803); PnrtA; PntcA; PppsA (e.g., fromCyanobacteria ABICyano1 (SEQ ID NO: 9)); PpsaA; PpsbD; PpstS (e.g., fromCyanobacteria ABICyano1 (SEQ ID NO: 10); PrbcL (e.g., from Synechocystissp. PCC6803); PrbcLS; PmpA (e.g., from Cyanobacteria ABICyano1 (SEQ IDNO: 11); PrpoA; PrpsL; PTac; Ptcr; PsbA2 (e.g., from SynechocystisPCC6803); PsigB; PsmtA (e.g., from Synechococcus sp. PCC 7002 andSynechococcus PCC 7942); and PziaA (e.g., from Synechocystis sp.PCC6803). Homologous promoters from other species (e.g., Synechococcuselongatus, Arthrospira maxima, Arthrospira platensis, and Cyanobacterumaponinum) as appropriate can also be used.

PhspA, Pc1pB1, and PhliB can be induced by heat shock (e.g., raising thegrowth temperature of the photosynthetic microorganism culture (theculture) from 300° C. to 400° C.), cold shock (e.g., reducing the growthtemperature of the culture from 300° C. to 20° C.), oxidative stress(e.g., by adding oxidants such as hydrogen peroxide to the culture), orosmotic stress (e.g., by increasing the salinity of the culture). PsigBcan be induced by stationary growth, heat shock, and osmotic stress.PntcA and PnblA can be induced by decreasing the concentration ofnitrogen in the growth medium and PpsaA and PpsbA2 can be induced by lowlight or high light conditions. PhtpG can be induced by osmotic stressand heat shock. PcrhC can be induced by cold shock. An increase incopper concentration can be used to induce PpetE, whereas PpetJ isinduced by decreasing copper concentration. PaztA, PsmtA, and PziaA canbe induced by adding Zn²⁺. PnrsB can be induced by adding Ni²⁺. PcorTcan be induced by adding cobalt. Additional details of these promoterscan be found, for example, in PCT/EP2009/060526.

The Ptrc promoter is inducible by addition of the chemical IPTG. In theabsence of IPTG the promoter has relatively low level of activity and inthe presence of IPTG it has a relatively high level of activity. It isnot a strictly “on/off” promoter, however. For some genes the low levelof activity in the absence of IPTG is enough to cause a phenotype (e.g.expression of N-SrrA). For other genes the higher level of inducedactivity is required to cause a phenotype (e.g. expression of N-RpaB(see FIG. 4)). Thus, in particular embodiments, uninduced can meanconstitutive expression at a low level, such as N-SrrA expressionobserved in the absence of IPTG.

Useful constitutive or inducible promoters are also described in, forexample: Samartzidou et al., Plant Physiol., 1998; 117:225-234; Duran etal., J. of Biol. Chem., 2004; 279:7229-7233; Singh et al., ArchMicrobiol., 2006; 186:273-286; Imamura et al., FEBS Lett., 2003;554:357-362; Imamura et al., J. Biol. Chem., 2006; 281:2668-2675;Agrawal et al., Biochem. Biophys. Res. Commun., 1999; 255:47-53; Mohamedet al., Plant Mol. Biol., 1989; 13:693-700; Muramatsu et al., Plant CellPhysiol., 2006; 47:878-890; Marin et al., Plant Physiol., 2004;136:3290-3300; Marin et al., J. Bacteriol., 2002; 184:2870-2877; Qi etal., Appl. Environ. Microbiol., 2005; 71:5678-5684; Maeda et al., J.Bacteriol., 1998; 180:4080-4088; Herranen et al., Plant Cell Physiol.,2005; 46:1484-1493; Buikema et al., Proc. Natl. Acad. Sci. USA, 2001;98:2729-2734; Mary et al., Microbiol., 2004; 150:1271-1281; He et al.,J. Biol. Chem., 2001; 276:306-314; Fang et al., Curr. Microbiol., 2004;49:192-198; and Kappell et al., Arch. Microbiol., 2007; 187:337-342.

In the case that more than one coding sequence of interest is present,then, for example, the first and second coding sequence can becontrolled by one promoter thereby forming a transcriptional operon.Alternatively the first and second coding sequence can be operablylinked to different first and second promoters, respectively. When morethan one promoter is used, all can be constitutive promoters, all can beinducible promoters, or a combination of constitutive and induciblepromoters can be used.

Expression control can be tightened when mutations are introduced in theTATA-box, the operator sequence and/or the ribosomal binding site (RBS)of the promoter controlling the expression of the coding sequence sothat the promoter has at least 90% sequence identity to an endogenouspromoter of the modified photosynthetic microorganism. Examples of theseapproaches are described below in relation to promoters PnirA, PcorT andPsmtA.

In particular embodiments, PnirA can have the generalized nucleotidesequence of SEQ ID NO: 12 wherein each of the nucleotides n isindependently selected from: a, t, c and g and wherein the two (atg)s inthe 5′-region of the promoter are the start for NtcB binding sites, gtais the start for the NtcA binding site, ccg denotes the start of theRBS, and the 3′-atg is the start codon for the first recombinant codingsequence transcriptionally controlled by this promoter.

Another generalized DNA sequence of PnirA includes nucleotide changes inthe RBS leading to the generalized DNA sequence of SEQ ID NO: 13. Inparticular embodiments the modified PnirA can include changes in theoperator region (binding site for NtcB and NtcA) and the TATA boxleading to the generalized nucleotide sequence of SEQ ID NO: 14. Anothervariant of PnirA combines changes in the RBS, operator region and theTATA box to form SEQ ID NO: 15.

Particular embodiments provide the Co²⁺-inducible PcorT, which has thegeneral nucleotide sequence of SEQ ID NO: 16 wherein each of thenucleotides n is independently selected from: a, t, c and g and whereinthe 5′-cat is the start codon of corR (antisense orientation) and the3′-atg is the start codon for the first recombinant coding sequencetranscriptionally controlled by this promoter. A modified variant ofPcorT includes changes in the RBS having SEQ ID NO: 17. Another variantof PcorT includes changes in the TATA box having the general sequence ofSEQ ID NO: 18. A third modified PcorT combines the RBS and TATA boxmodifications into SEQ ID NO: 19.

Furthermore the Zn²⁺-inducible PsmtA from Synechococcus PCC 7002 can beused having the generalized nucleotide sequence of SEQ ID NO: 20.Changes in the RBS can lead to the following generalized nucleotidesequences of SEQ ID NO: 21 or SEQ ID NO: 22. Again, homologous sequencesfrom other species (e.g., Synechococcus elongatus, Arthrospira maxima,Arthrospira platensis, and Cyanobacterium aponinum) as appropriate mayalso be used.

As suggested, particular embodiments include codon optimization. Codonspreferred by a particular photosynthetic microorganism can be selectedto, for example, increase the rate of protein expression or to produce arecombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence. Such nucleotide sequences are typicallyreferred to as “codon-optimized.”

At least some of the nucleotide sequences to be expressed in modifiedphotosynthetic microorganisms can be codon-optimized for optimalexpression in a chosen Cyanobacterial strain. The underlying rationaleis that the codon usage frequency of highly expressed genes is generallycorrelated to the host cognate tRNA abundance. (Bulmer, Nature, 1987;325:728-730). In particular embodiments, the codon optimization is basedon the Cyanobacteria ABICyano1 (as well as its close relative species)codon usage frequency (host codon bias), in order to achieve desirableheterologous gene expression (Sharp et al., 1987; Nucleic Acids Res.,15:1281-1295). In particular embodiments, codon optimization can bebased on Synechococcus elongatus PCC 7942.

Codon optimization can be performed with the assistance of publiclyavailable software, such as Gene Designer (DNA 2.0). Additionalmodifications to minimize unwanted restriction sites, internalShine-Dalgarno sequences, and other sequences such as internaltermination sequences and repeat sequences can also be performed. Thesegeneral codon-optimization methods have been shown to result in up to1,000 fold higher expression of heterologous genes in target organisms(Welch et al., PLoS One 4, 2009; e7002; and Welch et al., J. of theRoyal Society, 2009; Interface 6 (Suppl 4):S467-S476.

In particular embodiments, a gene that has at least 85% sequenceidentity; 86% sequence identity; 87% sequence identity; 88% sequenceidentity; 89% sequence identity; 90% sequence identity; 91% sequenceidentity; 92% sequence identity; 93% sequence identity; 94% sequenceidentity; 95% sequence identity; 96% sequence identity; 97% sequenceidentity; 98% sequence identity; or 99% sequence identity to SEQ ID NO:23, SEQ ID NO: 33, SEQ ID NO: 41 and/or SEQ ID NO: 43 can be placedbehind a promoter in a neutral site to drive expression of N-RpaB,N-SrrA, guide RNA and/or Cas 9 respectively.

In particular embodiments, genes that encode a protein or guide RNAhaving 85% sequence identity; 86% sequence identity; 87% sequenceidentity; 88% sequence identity; 89% sequence identity; 90% sequenceidentity; 91% sequence identity; 92% sequence identity; 93% sequenceidentity; 94% sequence identity; 95% sequence identity; 96% sequenceidentity; 97% sequence identity; 98% sequence identity; or 99% sequenceidentity to SEQ ID NO: 2; SEQ ID NO: 34; SEQ ID NO: 42 or SEQ ID NO: 44,can be placed behind a promoter in a neutral site to drive expression ofN-RpaB, N-SrrA guide RNA or Cas9 variants.

Variants of N-RpaB, N-SrrA or Cas9 include proteins having one or moreamino acid additions, deletions, stop positions, or substitutions, ascompared to N-RpaB (SEQ ID NO: 2), N-SrrA (SEQ ID NO: 34) or Cas9 (SEQID NO: 44). Variants of N-RpaB N-SrrA and Cas9 have at least 85%sequence identity; 86% sequence identity; 87% sequence identity; 88%sequence identity; 89% sequence identity; 90% sequence identity; 91%sequence identity; 92% sequence identity; 93% sequence identity; 94%sequence identity; 95% sequence identity; 96% sequence identity; 97%sequence identity; 98% sequence identity; or 99% sequence identity toN-RpaB, N-SrrA or Cas9 and cause a statistically significant increase ina photosynthetic microorganism's photosynthetic capacity as compared toa photosynthetic microorganism that has not been modified to havereduced RpaB pathway activity. Variants of Cas9 cause a statisticallysignificant increase in a photosynthetic microorganism's photosyntheticcapacity as compared to a photosynthetic microorganism that has not beenmodified to have reduced RpaB pathway activity in combination with guideRNA or variants thereof.

An amino acid substitution of N-RpaB, N-SrrA, or Cas9 can be aconservative or a non-conservative substitution. A “conservativesubstitution” involves a substitution found in one of the followingconservative substitutions groups: Group 1: Alanine (Ala; A), Glycine(Gly; G), Serine (Ser; S), Threonine (Thr; T); Group 2: Aspartic acid(Asp; D), Glutamic acid (Glu; E); Group 3: Asparagine (Asn; N),Glutamine (Gln; Q); Group 4: Arginine (Arg; R), Lysine (Lys; K),Histidine (His; H); Group 5: Isoleucine (Ile; 1), Leucine (Leu; L),Methionine (Met; M), Valine (Val; V); and Group 6: Phenylalanine (Phe;F), Tyrosine (Tyr; Y), Tryptophan (Trp; W).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function, chemical structure, or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cys; acidic: Asp, Glu, Asn, and Gln; smallaliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, andGly; polar, negatively charged residues and their amides: Asp, Asn, Glu,and Gln; polar, positively charged residues: His, Arg, and Lys; largealiphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and largearomatic residues: Phe, Tyr, and Trp. As indicated, in particularembodiments, conservative substitutions can include substituting Asp56with Glu, Ser, Thr or Tyr.

Non-conservative substitutions include those that affect the function ofN-RpaB, N-SrrA or Cas9 in a statistically-significant manner.Non-conservative substitutions include those in which (i) a hydrophilicresidue (e.g. Ser or Thr) is substituted by a hydrophobic residue (e.g.Leu, Ile, Phe, Val, or Ala); (ii) a Cys or Pro is substituted by anyother residue; (iii) a residue having an electropositive side chain(e.g. Lys, Arg, or His) is substituted by an electronegative residue(e.g. Gln or Asp); or (iv) a residue having a bulky side chain (e.g.Phe), is substituted by one not having a bulky side chain, (e.g. Gly).In particular embodiments, non-conservative substitutions can be made atAsp56 in sequences having 90% or more sequence identity with wild-typeRpaB or at Asp64 in sequences having 90% or more sequence identity withwild-type SrrA to create decoys with non-functioning phospho-receiverdomains. Additional information is found in Creighton (1984) Proteins,W.H. Freeman and Company.

In particular embodiments, N-RpaB variants retain Asp at position 56. Inparticular embodiments, no variant positions are found at position 50,51, 52, 53, 54, 55, 56, 57, 58, 59 or 60. In particular embodiments,N-SrrA variants retain Asp at position 64. In particular embodiments, novariant positions are found at position 58, 59, 60, 61, 62, 63, 64, 65,66, 67 or 68.

Variants of guide RNA include RNA sequences having one or morenucleotide additions, deletions, stop positions, or substitutions, ascompared to SEQ ID NO: 42. Variants of guide RNA have at least 85%sequence identity; 86% sequence identity; 87% sequence identity; 88%sequence identity; 89% sequence identity; 90% sequence identity; 91%sequence identity; 92% sequence identity; 93% sequence identity; 94%sequence identity; 95% sequence identity; 96% sequence identity; 97%sequence identity; 98% sequence identity; or 99% sequence identity toSEQ ID NO: 42 and function with Cas9 or variants thereof to cause astatistically significant increase in a photosynthetic microorganism'sphotosynthetic capacity as compared to a photosynthetic microorganismthat has not been modified to have reduced RpaB pathway activity.

Variants incorporating stop positions can be biologically activefragments. Biologically active fragments have 0.1, 0.5, 1, 2, 5, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 150, 200, 300, 400, 500,600, 700, 800, 900, 1000% or more of the activity of a referencesequence. A reference sequence refers generally to an amino acidsequence, RNA sequence, or a nucleic acid coding sequence expressing aprotein and/or guide RNA that reduces RpaB pathway activity as describedherein.

“% sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweensequences as determined by the match between strings of such sequences.“Identity” (often referred to as “similarity”) can be readily calculatedby known methods, including those described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, NY (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994);Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Oxford University Press, NY (1992). Preferred methods todetermine sequence identity are designed to give the best match betweenthe sequences tested. Methods to determine sequence identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR, Inc., Madison, Wis.). Multiple alignment of thesequences can also be performed using the Clustal method of alignment(Higgins and Sharp, CAB/OS, 1989; 5:151-153 with default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include theGCG suite of programs (Wisconsin Package Version 9.0, Genetics ComputerGroup (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul et al., J.Mol. Biol., 1990; 215:403-410; DNASTAR (DNASTAR, Inc., Madison, Wis.);and the FASTA program incorporating the Smith-Waterman algorithm(Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994),Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher Plenum,New York, N.Y.). Within the context of this disclosure it will beunderstood that where sequence analysis software is used for analysis,the results of the analysis are based on the “default values” of theprogram referenced. As used herein “default values” will mean any set ofvalues or parameters which originally load with the software when firstinitialized.

Insertion (e.g., transformation) of a nucleotide sequence (e.g., avector) into a photosynthetic microorganism can be achieved using anyappropriate method including, for example, natural transformation (e.g.,natural DNA uptake; see, e.g., Chung et al., FEMS Microbiol. Lett.,1998; 164: 353-361; Frigaard et al., Methods Mol. Biol., 2004;274:325-40; Zang et al., J. Microbiol., 2007; 45:241-245); conjugation(e.g., bi- or tri-parental mating), transduction, glass beadtransformation (see, e.g., Kindle et al., J. Cell Biol., 1989;109:2589-601; Feng et al., Mol. Biol. Rep., 2009; 36:1433-9; U.S. Pat.No. 5,661,017), silicon carbide whisker transformation (see, e.g.,Dunahay et al., Methods Mol. Biol., 1997; 62: 503-9), biolistics (see,e.g., Dawson et al., Curr. Microbiol., 1997; 35:356-62; Hallmann et al.,Proc. Natl. Acad. USA, 1997; 94:7469-7474; Doestch et al., Curr. Genet.,2001; 39:49-60; Jakobiak et al., Protist, 2004; 155:381-93; Ramesh etal., Methods Mol. Biol., 2004; 274: 355-307; Tan et al., J. Microbiol.,2005; 43:361-365; Steinbrenner et al., Appl Environ. Microbiol., 2006;72:7477-7484; Kroth, Methods Mol. Biol., 2007; 390:257-267; U.S. Pat.No. 5,661,017); electroporation (see, e.g., Kjaerulff et al.,Photosynth. Res., 1994; 41:277-283; Iwai et al., Plant Cell Physiol.,2004; 45:171-5; Ravindran et al., J. Microbiol. Methods, 2006; 66:174-6;Sun et al., Gene, 2006; 377: 140-149; Wang et al., Appl. Microbiol.Biotechnol., 2007; 76:651-657; Chaurasia et al., J. Microbiol. Methods,2008; 73:133-141; Ludwig et al., Appl. Microbiol. Biotechnol., 2008;78:729-35), laser-mediated transformation, or incubation with DNA in thepresence of or after pre-treatment with any of poly(amidoamine)dendrimers (see, e.g., Pasupathy et al., J. Biotechnol., 2008;3:1078-82), polyethylene glycol (see, e.g., Ohnuma et al., Plant CellPhysiol., 2008; 49:117-120), cationic lipids (see, e.g., Muradawa etal., J. Biosci. Bioeng., 2008; 105: 77-80), dextran, calcium phosphate,or calcium chloride (see, e.g., Mendez-Alvarez et al., J. Bacteriol.,1994; 176:7395-7397), optionally after treatment of the cells with cellwall-degrading enzymes (see, e.g., Perrone et al., Mol. Biol. Cell,1998; 9:3351-3365).

In addition, the vector can be modified to allow for integration into achromosome by adding an appropriate DNA sequence homologous to thetarget region of the photosynthetic microorganism genome, or through invivo transposition by introducing the mosaic ends (ME) to the vector.Once a plasmid is established in a photosynthetic microorganism, it canbe present, for example, at a range of from 1 to many copies per cell.

Insertion methods described above can be used for introducing nucleotidesequences (e.g., vectors) into Cyanobacterial cells harboring anextracellular polymer layer (EPS). Non-limiting examples forCyanobacteria with an EPS include several Nostoc and Anabaena strains,such as Nostoc commune, and Anabanena cylindrica and several Cyanothecesp. strains, such as Cyanothece PCC9224, Cyanothece CA 3, Cyanothece CE4, Cyanothece ET5, Cyanothece ET 2, and Cyanospira capsulata ATCC 43193.Further examples of Cyanobacteria with an EPS include Aphanocapsa,Cyanobacterium, Anacystis, Chroococcus, Gloeothece, Microcystis,Synechocystis, Lyngbya, Microcoleus, Oscillatora, Phormidium,Arthrospira, Anabaena, Cyanospira, Nostoc, Scytonema, Tolypothrix,Chlorogloeopsis, Fischerella, and Mastigocladus (see for example: DePhilippis et al., J. of Applied Phycology, 2001; 13:293-299; DePhilippis et al., FEMS Microbiol. Reviews, 1998; 22:151-175).

In Cyanobacteria, restriction systems can create barriers to theintroduction of exogenous nucleotide sequences. Restriction systemsinclude a restriction enzyme and a specific DNA methyltransferase.Specific methylation of the restriction enzyme recognition sequenceprotects DNA in the photosynthetic microorganism from degradation by thecorresponding restriction enzyme. Knowledge of particular restrictionsystems within particular bacterial cell types can allow one to protectexogenous nucleotide sequences by methylating it at particular sites toprevent degradation by the photosynthetic microorganism's restrictionsystem restriction enzyme(s). Thus, an understanding of theserestriction systems can be helpful in choosing appropriatetransformation protocols for particular bacteria. Particular restrictionsystems for different Cyanobacterial cells can be found atrebase.neb.com.

Nucleotide sequences used herein can include selectable markers toidentify modified photosynthetic microorganisms. Selectable markers canbe any identifying factor, usually an antibiotic or chemical resistancegene, that is able to be selected for based upon the marker gene'seffect, such as resistance to an antibiotic, resistance to a herbicide,colorimetric markers, enzymes, fluorescent markers, and the like,wherein the effect is used to track the transformation of a nucleotidesequence of interest and/or to identify a modified photosyntheticmicroorganism that has inherited the nucleotide sequence of interest.Examples of selectable marker genes known and used in the art include:genes providing resistance to ampicillin, gentamycin, hygromycin,kanamycin, spectinomycin, streptomycin, fluorescent proteins (e.g., fromPromega Corporation, Invitrogen, Clontech, Stratagene, BD BiosciencesPharmingen, Evrogen JSC), and the like.

Modified photosynthetic microorganisms, including Cyanobacteria, can becultured or cultivated according to techniques known in the art, such asthose described in Acreman et al., J. of Industrial Microbiol. andBiotechnol., 1994; 13:193-194), in addition to photobioreactor basedtechniques, such as those described in Nedbal et al., Biotechnol.Bioeng., 2008; 100:902-10. One example of typical laboratory cultureconditions for Cyanobacteria is growth in BG-11 medium (ATCC Medium 616)at 30° C. in a vented culture flask with constant agitation and constantillumination at 30-100 μmole photons m⁻² sec⁻¹.

Additional media for culturing Cyanobacteria, include Aiba and Ogawa(AO) Medium, Allen and Amon Medium plus Nitrate (ATCC Medium 1142),Antia's (ANT) Medium, Aquil Medium, Ashbey's Nitrogen-free Agar, ASN-IIIMedium, ASP 2 Medium, ASW Medium (Artificial Seawater and derivatives),ATCC Medium 617 (BG-11 for Marine Blue-Green Algae; Modified ATCC Medium616 [BG-11 medium]), ATCC Medium 819 (Blue-green Nitrogen-fixing Medium;ATCC Medium 616 [BG-11 medium] without NO₃), ATCC Medium 854 (ATCCMedium 616 [BG-11 medium] with Vitamin B₁₂), ATCC Medium 1047 (ATCCMedium 957 [MN marine medium] with Vitamin B₁₂), ATCC Medium 1077(Nitrogen-fixing marine medium; ATCC Medium 957 [MN marine medium]without NO₃), ATCC Medium 1234 (BG-11 Uracil medium; ATCC Medium 616[BG-11 medium] with uracil), Beggiatoa Medium (ATCC Medium 138),Beggiatoa Medium 2 (ATCC Medium 1193), BG-11 Medium for Blue Green Algae(ATCC Medium 616), Blue-Green (BG) Medium, Bold's Basal (BB) Medium,Castenholtz D Medium, Castenholtz D Medium Modified (HalophilicCyanobacteria), Castenholtz DG Medium, Castenholtz DGN Medium,Castenholtz ND Medium, Chlorofexus Broth, Chlorofexus Medium (ATCCMedium 920), Chu's #10 Medium (ATCC Medium 341), Chu's #10 MediumModified, Chu's #11 Medium Modified, DCM Medium, DYIV Medium, E27Medium, E31 Medium and Derivatives, f/2 Medium, f/2 Medium Derivatives,Fraquil Medium (Freshwater Trace Metal-Buffered Medium), Gorham's Mediumfor Algae (ATCC Medium 625), h/2 Medium, Jaworski's (JM) Medium, KMedium, L1 Medium and Derivatives, MN Marine Medium (ATCC Medium 957),Plymouth Erdschreiber (PE) Medium, Prochlorococcus PC Medium, ProteosePeptone (PP) Medium, Prov Medium, Prov Medium Derivatives, S77 plusVitamins Medium, S88 plus Vitamins Medium, Saltwater Nutrient Agar (SNA)Medium and Derivatives, SES Medium, SN Medium, Modified SN Medium, SNAXMedium, Soil/Water Biphasic (S/W) Medium and Derivatives, SOT Medium forArthrospira (Spirulina): ATCC Medium 1679, Spirulina (SP) Medium, vanRijn and Cohen (RC) Medium, Walsby's Medium, Yopp Medium, and Z8 Medium,among others.

Particular embodiments disclosed herein demonstrate increasedphotoautotrophic growth of photosynthetic microorganisms with reducedRpaB pathway activity. The advantage of Rpa-B down regulation onphotoautotrophic growth is evident shortly after culturing begins withthe increased growth becoming more apparent as the culture is propagatedover time. See, for example, FIG. 5. In particular embodiments, a volumefraction of the liquid culture can be replaced with an equivalent volumeof new growth media during the culturing period, as is understood by oneof ordinary skill in the art.

Modified photosynthetic microorganisms disclosed herein have increasedphotosynthetic capacity. Photosynthetic capacity is defined here as themaximum rate of electron transport through the photosynthetic electrontransport chain. Photosynthetic capacity can be independently measuredfor different segments of the photosynthetic electron transport chain.For the segment through PSII, photosynthetic capacity can be measured bydetermining the rate of oxygen evolution of whole cells in the presenceof para-benzoquinone and potassium ferricyanide, which serve to acceptelectrons directly from PSII, allowing for PSII oxygen evolution to runat its maximal rate, independent of down-stream proteins in the electrontransport chain. For the segment including the entire electron transportchain through PSI, increased photosynthetic capacity can be measured bydetermining the rate of oxygen uptake of whole cells in the presence ofmethyl viologen and potassium cyanide, which serve to accept electronsdirectly from PSI, allowing for the entire electron transport chain torun at maximal rate, independent of down-stream proteins in, e.g.,carbon fixation or nitrate reduction. In this latter embodiment, theuptake rate of oxygen is a measure of the photosynthetic capacitybecause of the specific chemistry of the assay involving methylviologen, which follows the following half reactions:2H₂O→4H⁺+4e ⁻+O₂2O₂+4H⁺+4e ⁻→2H₂O₂This leads to a balanced equation below, where 1 molecule of oxygen isconsumed for every molecule of O₂ that could potentially be evolved:2H₂O+O₂→2H₂O₂Increased photosynthetic capacity can increase total carbon fixation,production of carbon containing compounds, and growth (biomassaccumulation).

Particular embodiments include increasing photosynthetic biomassaccumulation from photoautotrophic growth of a photosyntheticmicroorganism comprising modifying the photosynthetic microorganism todown-regulate RpaB pathway activity within the photosyntheticmicroorganism as compared to photosynthetic biomass accumulation of thesame species. See, for example, FIG. 6.

The Exemplary Embodiments below describe particular embodiments of thedisclosure. Those of ordinary skill in the art should recognize in lightof the present disclosure that many changes can be made to the specificembodiments disclosed herein and still obtain alike or similar resultwithout departing from the spirit and scope of the disclosure.

Exemplary Embodiments

1. A modified photosynthetic microorganism with increased photosyntheticcapacity as compared to a photosynthetic microorganism of the samespecies without the modification.

2. A modified photosynthetic microorganism of embodiment 1 wherein themodified photosynthetic microorganism includes a genetic modification.

3. A modified photosynthetic microorganism of embodiment 1 or agenetically-modified photosynthetic microorganism of embodiment 2wherein the increased photosynthetic capacity results from decreasedRpaB pathway activity.

4. A genetically-modified photosynthetic microorganism of embodiment 3wherein the decreased RpaB pathway activity results from (i) expressionof at least one exogenous nucleotide sequence and/or (ii) a wild-typenucleotide sequence deletion wherein (i) and/or (ii) decreases RpaBpathway activity as compared to a photosynthetic microorganism of thesame species without the modification(s).5. A genetically-modified photosynthetic microorganism of embodiment 4wherein the exogenous nucleotide sequence (i) results in translation ofan incomplete or unstable RpaB protein; (ii) results in translation ofan RpaB protein that folds incorrectly; (iii) reduces transcription ofthe RpaB gene; (iv) results in incomplete transcription of the RpaBgene; (v) interferes with an encoded RpaB RNA transcript and/or (vi)reduces translation of RpaB.6. A genetically-modified photosynthetic microorganism of embodiment 4wherein the exogenous nucleotide sequence is a foreign set of base pairsinserted or substituted into the RpaB coding region.7. A genetically-modified photosynthetic microorganism of embodiment 4wherein the exogenous nucleotide sequence is an antisense sequence thatinterferes with transcription or translation of the RpaB gene.8. A genetically-modified photosynthetic microorganism of embodiment 4wherein the exogenous nucleotide sequence expresses an RpaB decoy underthe control of a promoter.9. A genetically-modified photosynthetic microorganism of any of thepreceding embodiments including at least two of the described exogenousnucleotide sequences, at least three of the described exogenousnucleotide sequences, at least four of the described exogenousnucleotide sequences, or at least five of the described exogenousnucleotide sequences.10. A genetically-modified photosynthetic microorganism of embodiments 8or 9 wherein the RpaB decoy is an N-terminal fragment of RpaB or SrrAincluding a phosphor-receiver domain but no DNA binding domain or anon-functional DNA binding domain.11. A genetically-modified photosynthetic microorganism of any ofembodiments 8-10 wherein the RpaB decoy is a fragment of wild type RpaBincluding Asp56 or a conservative substitution thereof or an N-terminalfragment of wild type SrrA including Asp64 or a conservativesubstitution thereof.12. A genetically-modified photosynthetic microorganism of any ofembodiments 8-10 wherein the RpaB decoy is N-RpaB or N-SrrA.13. A genetically-modified photosynthetic microorganism of any ofembodiments 8-10 wherein the RpaB decoy is an N-RpaB variant thatmaintains Asp56 or a conservative substitution thereof or an N-SrrAvariant that maintains Asp64 or a conservative substitution thereof.14. A genetically-modified photosynthetic microorganism of any ofembodiments 4-13 wherein the exogenous nucleotide sequence includes SEQID NO: 23 or SEQ ID NO: 33.15. A genetically-modified photosynthetic microorganism of any ofembodiments 4-13 wherein the exogenous nucleotide sequence includes SEQID NO: 23 and SEQ ID NO: 33.16. A genetically-modified photosynthetic microorganism of any ofembodiments 4-15 wherein the exogenous nucleotide sequence expressesguide RNA and a Cas9 protein.17. A genetically-modified photosynthetic microorganism of any ofembodiments 4-16 wherein the exogenous nucleotide sequence includes SEQID NO: 41.18. A genetically-modified photosynthetic microorganism of any ofembodiments 4-17 wherein the exogenous nucleotide sequence includes SEQID NO: 43.19. A genetically-modified photosynthetic microorganism of any ofembodiments 4-18 wherein the exogenous nucleotide sequence includes SEQID NO: 41 and SEQ ID NO: 43.20. A genetically-modified photosynthetic microorganism of embodiment 16wherein the guide RNA includes SEQ ID NO: 42 or a biologically activefragment thereof.21. A genetically-modified photosynthetic microorganism of embodiment 16or 20 wherein the Cas9 protein includes SEQ ID NO: 44 or a variantthereof including a conservative substitution.22. A genetically-modified photosynthetic microorganism of embodiment 16or 20 wherein the Cas9 protein includes a substitution at amino acidpositions 10 and/or 841.23. A genetically-modified photosynthetic microorganism of any ofembodiments 8-22 wherein the promoter is an inducible promoter.24. A genetically-modified photosynthetic microorganism of any ofembodiments 8-22 wherein the promoter is endogenous to the genome of thegenetically-modified photosynthetic microorganism.25. A genetically-modified photosynthetic microorganism of any ofembodiments 1-24 wherein the genetically-modified photosyntheticmicroorganism is a Cyanobacteria.26. A genetically-modified photosynthetic microorganism of any ofembodiments 1-24 wherein the genetically-modified photosyntheticmicroorganism is a Cyanobacteria selected from Synechococcus elongatus,Arthrospira maxima, Arthrospira platensis, and Cyanobacterum aponinum.27. A genetically-modified photosynthetic microorganism of any ofembodiments 1-26 wherein the genetically-modified photosyntheticmicroorganism has increased photosynthetic capacity as compared to awild type photosynthetic microorganism of the same species.28. A method for increasing photosynthetic capacity and/or biomassaccumulation of a photosynthetic microorganism including modifying thephotosynthetic microorganism to reduce RpaB pathway activity within thephotosynthetic microorganism as compared to a photosyntheticmicroorganism of the same species without the modification or ascompared to a wild type photosynthetic microorganism of the samespecies.29. A method of embodiment 28 wherein the modifying includes geneticallymodifying the photosynthetic microorganism.30. A method of embodiment 28 or 29 wherein the modifying includesinserting an exogenous nucleotide sequence into the photosyntheticmicroorganism or deleting an endogenous nucleotide sequence from thephotosynthetic microorganism.31. A method of embodiment 30 wherein the exogenous nucleotide sequence(i) results in translation of an incomplete or unstable RpaB protein;(ii) results in translation of an RpaB protein that folds incorrectly;(iii) reduces transcription of the RpaB gene; (iv) results in incompletetranscription of the RpaB gene; (v) interferes with an encoded RpaB RNAtranscript and/or (vi) reduces translation of RpaB.32. A method of any of embodiments 28-31 wherein modifying includesinserting or substituting a foreign set of base pairs into the RpaBcoding region.33. A method of any of embodiments 28-32 wherein modifying includesinserting an antisense sequence that interferes with transcription ortranslation of the RpaB gene.34. A method of any of embodiments 28-33 wherein the modifying includesinserting an exogenous nucleotide sequence that expresses an RpaB decoyunder the control of a promoter into the photosynthetic microorganism.35. A method of embodiment 34 wherein the RpaB decoy is an N-terminalfragment of RpaB or SrrA including a phosphor-receiver domain but no DNAbinding domain or a non-functional DNA binding domain.36. A method of embodiment 34 or 35 wherein the expressed RpaB decoy isan N-terminal fragment of wild type RpaB including Asp56 or aconservative substitution thereof an N-terminal fragment of wild typeSrrA including Asp64 or a conservative substitution thereof.37. A method of embodiment 34 or 35 wherein the expressed RpaB decoy isN-RpaB or N-SrrA.38. A method of embodiment 34 or 35 wherein the expressed RpaB decoy isan N-RpaB variant that maintains Asp56 or a conservative substitutionthereof or an N-SrrA variant that maintains Asp64 or a conservativesubstitution thereof.39. A method of embodiment 34 wherein the exogenous nucleotide sequenceincludes SEQ ID NO: 23 or SEQ ID NO: 33.40. A method of embodiment 34 wherein the exogenous nucleotide sequenceincludes SEQ ID NO: 23 and SEQ ID NO: 33.41. A method of any of embodiments 28-40 wherein the exogenousnucleotide sequence expresses guide RNA and a Cas9 protein.42. A method of embodiment 41 wherein the exogenous nucleotide sequenceincludes SEQ ID NO: 41.43. A method of embodiment 41 or 42 wherein the exogenous nucleotidesequence includes SEQ ID NO: 43.44. A method of any of embodiments 41-43 wherein the exogenousnucleotide sequence includes SEQ ID NO: 41 and SEQ ID NO: 43.45. A method of any of embodiments 41-44 wherein the guide RNA includesSEQ ID NO: 42 or a biologically active fragment thereof.46. A method of any of embodiments 41-45 wherein the Cas9 proteinincludes SEQ ID NO: 44 or a variant thereof including a conservativesubstitution.47. A method of any of embodiments 41-46 wherein the Cas9 proteinincludes a substitution at amino acid positions 10 and/or 841.48. A method of any of embodiments 34-47 wherein the promoter is aninducible promoter.49. A method of any of embodiments 34-48 wherein the promoter isendogenous to the genome of the genetically-modified photosyntheticmicroorganism.50. A method of any of embodiments 28-49 wherein thegenetically-modified photosynthetic microorganism is a Cyanobacteria.51. A method of any of embodiments 28-50 wherein thegenetically-modified photosynthetic microorganism is a Cyanobacteriaselected from Synechococcus elongatus, Arthrospira maxima, Arthrospiraplatensis, and Cyanobacterium aponinum.52. A method of any of embodiments 28-51 wherein thegenetically-modified photosynthetic microorganism has increasedphotosynthetic capacity as compared to a photosynthetic microorganism ofthe same species without the modification or as compared to a wild typephotosynthetic microorganism of the same species.53. A method of any of embodiments 28-52 wherein thegenetically-modified photosynthetic microorganism shows increasedphotosynthetic biomass accumulation from photoautotrophic growth ascompared to a photosynthetic microorganism of the same species withoutthe modification or as compared to a wild type photosyntheticmicroorganism of the same species.54. A method of increasing photoautotrophic growth of a photosyntheticmicroorganism including

modifying the photosynthetic microorganism to reduce RpaB pathwayactivity within the photosynthetic microorganism as compared to aphotosynthetic microorganism of the same species without themodification or as compared to a wild type photosynthetic microorganismof the same species and culturing the modified photosyntheticmicroorganism in a liquid culture, thereby increasing photoautotrophicgrowth of the photosynthetic microorganism as compared to thephotosynthetic microorganism of the same species without themodification or as compared to the wild type photosyntheticmicroorganism of the same species.

55. A method of embodiment 54 wherein the modifying includes geneticallymodifying the photosynthetic microorganism.

56. A method of embodiment 54 or 55 wherein the modifying includesinserting an exogenous nucleotide sequence into the photosyntheticmicroorganism or deleting an endogenous nucleotide sequence from thephotosynthetic microorganism.

57. A method of embodiment 56 wherein the exogenous nucleotide sequence(i) results in translation of an incomplete or unstable RpaB protein;(ii) results in translation of an RpaB protein that folds incorrectly;(iii) reduces transcription of the RpaB gene; (iv) results in incompletetranscription of the RpaB gene; (v) interferes with an encoded RpaB RNAtranscript and/or (vi) reduces translation of RpaB.58. A method of any of embodiments 54-57 wherein modifying includesinserting or substituting a foreign set of base pairs into the RpaBcoding region.59. A method of any of embodiments 54-58 wherein modifying includesinserting an antisense sequence that interferes with transcription ortranslation of the RpaB gene.60. A method of any of embodiments 54-59 wherein the modifying includesinserting an exogenous nucleotide sequence that expresses an RpaB decoyunder the control of a promoter into the photosynthetic microorganism.61. A method of embodiment 60 wherein the RpaB decoy is an N-terminalfragment of RpaB or SrrA including a phosphor-receiver domain but no DNAbinding domain or a non-functional DNA binding domain.62. A method of embodiment 60 or 61 wherein the expressed RpaB decoy isan N-terminal fragment of wild type RpaB including Asp56 or aconservative substitution thereof an N-terminal fragment of wild typeSrrA including Asp64 or a conservative substitution thereof.63. A method of embodiment 60 or 61 wherein the expressed RpaB decoy isN-RpaB or N-SrrA.64. A method of embodiment 60 or 61 wherein the expressed RpaB decoy isan N-RpaB variant that maintains Asp56 or a conservative substitutionthereof or an N-SrrA variant that maintains Asp64 or a conservativesubstitution thereof.65. A method of embodiment 60 wherein the exogenous nucleotide sequenceincludes SEQ ID NO: 23 or SEQ ID NO: 33.66. A method of embodiment 60 wherein the exogenous nucleotide sequenceincludes SEQ ID NO: 23 and SEQ ID NO: 33.67. A method of any of embodiments 54-66 wherein the exogenousnucleotide sequence expresses guide RNA and a Cas9 protein.68. A method of embodiment 67 wherein the exogenous nucleotide sequenceincludes SEQ ID NO: 41.69. A method of embodiment 67 or 68 wherein the exogenous nucleotidesequence includes SEQ ID NO: 43.70. A method of any of embodiments 67-69 wherein the exogenousnucleotide sequence includes SEQ ID NO: 41 and SEQ ID NO: 43.71. A method of any of embodiments 67-70 wherein the guide RNA includesSEQ ID NO: 42 or a biologically active fragment thereof.72. A method of any of embodiments 67-71 wherein the Cas9 proteinincludes SEQ ID NO: 44 or a variant thereof including a conservativesubstitution.73. A method of any of embodiments 67-72 wherein the Cas9 proteinincludes a substitution at amino acid positions 10 and/or 841.74. A method of any of embodiments 60-73 wherein the promoter is aninducible promoter.75. A method of any of embodiments 60-74 wherein the promoter isendogenous to the genome of the genetically-modified photosyntheticmicroorganism.76. A method of any of embodiments 54-75 wherein thegenetically-modified photosynthetic microorganism is a Cyanobacteria.77. A method of any of embodiments 54-76 wherein thegenetically-modified photosynthetic microorganism is a Cyanobacteriaselected from Synechococcus elongatus, Arthrospira maxima, Arthrospiraplatensis, and Cyanobacterium aponinum.

When an inserted exogenous promoter is endogenous to the genome of thegenetically-modified photosynthetic microorganism, this means that anextra copy of a wild-type promoter of the species is inserted as part ofa genetic construct.

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of orconsist of its particular stated element, step, ingredient or component.Thus, the terms “include” or “including” should be interpreted torecite: “comprise, consist of, or consist essentially of.” As usedherein, the transition term “comprise” or “comprises” means includes,but is not limited to, and allows for the inclusion of unspecifiedelements, steps, ingredients, or components, even in major amounts. Thetransitional phrase “consisting of” excludes any element, step,ingredient or component not specified. The transition phrase “consistingessentially of” limits the scope of the embodiment to the specifiedelements, steps, ingredients or components and to those that do notmaterially affect the embodiment. As used herein, a material effectwould cause a statistically-significant reduction in a modifiedCyanobacteria's increased photosynthetic capacity.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; 9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; 3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or dearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, if references have been made to patents, printedpublications, journal articles and other written text throughout thisspecification (referenced materials herein), each of the referencedmaterials are individually incorporated herein by reference in theirentirety for their referenced teaching.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the following examples or when applicationof the meaning renders any construction meaningless or essentiallymeaningless. In cases where the construction of the term would render itmeaningless or essentially meaningless, the definition should be takenfrom Webster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

What is claimed is:
 1. A genetically-modified Cyanobacterium comprisingan exogenous nucleotide sequence comprising SEQ ID NO: 33 that expressesan N-terminal fragment of SrrA polypeptide under the control of apromoter that results in down-regulated RpaB pathway activity in thegenetically-modified Cyanobacterium as compared to a Cyanobacterium ofthe same species without the exogenous nucleotide sequence, wherein theN-terminal fragment of the SrrA polypeptide comprises aphosphor-receiver domain but does not comprise a DNA binding domain or afunctional DNA binding domain.
 2. The genetically-modifiedCyanobacterium of claim 1, wherein the promoter is an inducible promoteror is endogenous to the genome of the genetically-modifiedCyanobacterium.
 3. The genetically-modified Cyanobacterium of claim 1,wherein the genetically-modified Cyanobacterium is a Cyanobacteriumselected from Synechococcus elongatus, Arthrospira maxima, Arthrospiraplatensis, and Cyanobacterium aponinum.
 4. The genetically-modifiedCyanobacterium of claim 1, wherein the genetically-modifiedCyanobacterium has increased (a) photosynthetic capacity as compared toa Cyanobacterium of the same species without the exogenous nucleotidesequence; and/or (b) photosynthetic biomass accumulation fromphotoautotrophic growth as compared to a Cyanobacterium of the samespecies without the exogenous nucleotide sequence.
 5. A method ofincreasing photosynthetic capacity and/or biomass accumulation of aCyanobacterium comprising modifying the Cyanobacterium to down-regulateRpaB pathway activity within the Cyanobacterium as compared to aCyanobacterium of the same species without the modification, wherein themodifying comprises inserting an exogenous nucleotide sequencecomprising SEQ ID NO: 33 that expresses an N-terminal fragment of SrrApolypeptide under the control of a promoter, wherein the N-terminalfragment of the SrrA polypeptide comprises a phosphor-receiver domainbut does not comprise a DNA binding domain or a functional DNA bindingdomain.
 6. The method of claim 5, wherein the promoter is an induciblepromoter or is endogenous to the genome of the genetically-modifiedCyanobacterium.
 7. The method of claim 5, wherein thegenetically-modified Cyanobacterium is a Cyanobacterium selected fromSynechococcus elongatus, Arthrospira maxima, Arthrospira platensis, andCyanobacterium aponinum.
 8. The method of claim 5, wherein thegenetically-modified Cyanobacterium has increased (a) photosyntheticcapacity as compared to a Cyanobacterium of the same species without theexogenous nucleotide sequence; and/or (b) photosynthetic biomassaccumulation from photoautotrophic growth as compared to aCyanobacterium of the same species without the exogenous nucleotidesequence.